Project Summary Breakdown of the inner endothelial blood-retinal barrier (BRB) is a major cause of retinal edema and resultant vision loss in vascular eye diseases. In addition to paracellular transport though the tight junctions between endothelial cells (ECs), transportation of substance, particularly large molecules, also occurs through transcellular vesicles (transcytosis) across ECs. Normal healthy blood vessels in the central nervous system, including retinas, display low rates of transcytosis essential for maintaining blood-brain barrier (BBB) and inner BRB. Yet molecular mechanisms governing retinal EC transcytosis are poorly understood, which considerably limits our ability to manipulate inner BRB to treat vascular eye disease. In this project we identified that Wnt signaling pathway, a pathway fundamentally important for angiogenesis and vascular integrity control, may act as a critical negative regulator of EC transcytosis to maintain inner BRB. Mutations in the inter-related Wnt signaling pathway involving the ligand Norrin, and the receptor Frizzled4 and co-receptor low density lipoprotein receptor-related protein 5 (LRP5) are linked with development of familial exudative vitreoretinopathy (FEVR) and Norrie disease, both with inner endothelial BRB breakdown. Activation of canonical Wnt signaling involves stabilization of ?-catenin, which then translocates to the nucleus to bind nuclear T-cell factor /lymphoid enhancer factor (TCF/LEF) to influence target genes. Our preliminary data show that loss of Wnt signaling in mouse models of FEVR and Norrie disease (Lrp5-/- and Norriny/- mice) significantly increased EC transcytosis, with decreased levels of MFSD2a (major facilitator super family domain containing 2a), a membrane transporter protein that suppresses EC transcytosis. We hypothesize that Wnt signaling is essential for maintaining a low transcytosis rate in retinal EC critical for inner BRB integrity, through Mfsd2a-dependent caveola vesicle modulation. We will test this hypothesis with three aims. In Aim 1 we will determine whether Wnt signaling controls EC transcytosis by measuring vascular permeability of both small and large molecules in Lrp5-/- and Norriny/- retinas, as well as in EC culture using transcytosis assay with Wnt modulation. In Aim 2 we will determine whether Wnt signaling regulates EC transcytosis through transcriptional regulation of MFSD2a, a transcytotic inhibitor, using a combination of ex vivo and in vitro approaches. In Aim 3, we will delineate whether Wnt signaling and MFSD2a control MFSD2a-assisted caveolar vesicle formation, transportation, and exocytosis. This proposed work will uncover novel fundamental molecular mechanisms governing EC transcytosis and inner BRB integrity, which is of significance in aiding development of improved strategies to manipulate inner BRB. Findings from this work will also be highly relevant for modulation of BBB in the central nervous system, as well as development of drug delivery methods through BBB and inner BRB.